*Target (protein/gene name): Ricin *NCBI Gene # : LOC8261245 *Protein ID: 2AAI *Organism (including strain): Ricinus communis (castor bean) Etiologic Risk Group (see link below): BSL1 */Disease Information (sort of like the Intro to your Mini Research Write up):
Ricin is a protein produced by the plant Ricinius communis (Castor bean) and is one of the deadliest chemical compounds in the world with LD50 as low as 1-2ug/kg if injected, 21-43ug/kg if inhaled, and 1-20mg/kg if ingested. The plant is native to Africa but found all over the world due to its industrial, cosmetic, and ornamental uses. It produces Ricin in it's leaves and particularly in its seeds which are used to make castor oil a particular common oil (Ricin is water soluble and does not make it to the oil). Due to it's availability it has been used in multiple murders and is a likely biological weapon. It's symptoms begin to show at different times depending on the method of uptake anywhere from 3-36 hours. Symptoms include: bleeding, diarrhea, vomiting, abdominal pain, low blood pressure, and dehydration. It is a 2 chain protein with B allowing access to the cell and A causing symptoms. Chain A inhibits ribosomes by changing the nucleotide base in the ribosome whether in the ribosome or by changing the rRNA in a highly conserved sequence. The simple nature of the change allows it to inhibit 1500 ribosomes a minute. [1] Chain B binds to surface carbohydrate receptors while maintaining normal function of the receptors as well. Applications of better understanding Ricin are not limited to bioweapon prevention but potential targeting of cancer cell ribosomes.
Purification Method:
The clear lysate was precipitated with ammonium sulfate (40% saturation) and the pellet was dissolved and dialysed against Buffer A (10 mM Tris, pH 7.4, 100 mM KCl). The dialysate was filtered through a 0.45-pm filter and loaded onto a strong anion-exchange column Q Sepharose Fast Flow (2.6 X 10 cm), which had been preequilibrated with Buffer A. Unbound proteins were washed off the column with Buffer A and the bound fraction was eluted with Buffer B (Buffer A containing 1.0 M NaCl). Proteins eluted from the column as well as the unbound fraction were analyzed on a 12% SDSPAGE. Image of protein (PyMol with features delineated and shown separately): *Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
*length of your protein in Amino Acids 267 Molecular Weight: 58859.51 Molar Extinction coefficient of your protein at 280 nm wavelength: 89895 TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it. *CDS Gene Sequence (paste as text only):
*GC% Content for gene: *CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): *GC% Content for gene (codon optimized):
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): (link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol) -- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences): **
Ricin Toxin Chain A/B:
*Target (protein/gene name): Ricin*NCBI Gene # : LOC8261245
*Protein ID: 2AAI
*Organism (including strain): Ricinus communis (castor bean)
Etiologic Risk Group (see link below): BSL1
*/Disease Information (sort of like the Intro to your Mini Research Write up):
Ricin is a protein produced by the plant Ricinius communis (Castor bean) and is one of the deadliest chemical compounds in the world with LD50 as low as 1-2ug/kg if injected, 21-43ug/kg if inhaled, and 1-20mg/kg if ingested. The plant is native to Africa but found all over the world due to its industrial, cosmetic, and ornamental uses. It produces Ricin in it's leaves and particularly in its seeds which are used to make castor oil a particular common oil (Ricin is water soluble and does not make it to the oil). Due to it's availability it has been used in multiple murders and is a likely biological weapon. It's symptoms begin to show at different times depending on the method of uptake anywhere from 3-36 hours. Symptoms include: bleeding, diarrhea, vomiting, abdominal pain, low blood pressure, and dehydration. It is a 2 chain protein with B allowing access to the cell and A causing symptoms. Chain A inhibits ribosomes by changing the nucleotide base in the ribosome whether in the ribosome or by changing the rRNA in a highly conserved sequence. The simple nature of the change allows it to inhibit 1500 ribosomes a minute. [1] Chain B binds to surface carbohydrate receptors while maintaining normal function of the receptors as well. Applications of better understanding Ricin are not limited to bioweapon prevention but potential targeting of cancer cell ribosomes.
Link to TDR Targets page (if present): N/A
Link to Gene Database: https://www.ncbi.nlm.nih.gov/gene/8261245
Essentiality of this protein: Both chains are essential to function.
Complex of proteins?: Yes, chain A and B
Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism):
http://robertus.cm.utexas.edu/papers/rta-pta.pdf
http://www.rcsb.org/pdb/explore/literature.do?structureId=2AAI
https://pdb101.rcsb.org/motm/161
*EC#: 3.2.2.22
Link to BRENDA EC# page: http://www.brenda-enzymes.org/enzyme.php?ecno=3.2.2.22
-- Show screenshot of BRENDA enzyme mechanism schematic (?)
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4690108/ - assayable in multiple ways
Vector labs Chain A/B - $165/mg A&B $150/mg
Structure (PDB or Homology model)
-- PDB #: 2AAI
Current Inhibitors: No current treatment, developing inhibitors.
Expression Information (has it been expressed in bacterial cells): High levels can be produced and simply purified. As well as readily be recombined with Chain B to produce whole ricin
Purification Method:
The clear lysate was precipitated with ammonium sulfate (40% saturation) and the pellet was dissolved and dialysed against Buffer A (10 mM Tris, pH 7.4, 100 mM KCl). The dialysate was filtered through a 0.45-pm filter and loaded onto a strong anion-exchange column Q Sepharose Fast Flow (2.6 X 10 cm), which had been preequilibrated with Buffer A. Unbound proteins were washed off the column with Buffer A and the bound fraction was eluted with Buffer B (Buffer A containing 1.0 M NaCl). Proteins eluted from the column as well as the unbound fraction were analyzed on a 12% SDSPAGE.
Image of protein (PyMol with features delineated and shown separately):
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
*length of your protein in Amino Acids 267
Molecular Weight: 58859.51
Molar Extinction coefficient of your protein at 280 nm wavelength: 89895
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
*CDS Gene Sequence (paste as text only):
<span style="background-color: #ffffff; font-family: monospace,serif;">1 <span class="ff_line">tcatttcttt aaacttctta catttttttt tgtagcattc tttgtaagtg gaatgacaaa</span> 61 <span class="ff_line">accgttaatg atgttctttt aaaagtgaaa gatgtttata tattgcagta cagataatga</span> 121 <span class="ff_line">tatatctact gcactacata aaacaattta aatctccctg tttattttaa gaagttatat</span> 181 <span class="ff_line">tttctttctt tctcatccta agaaagttaa attactgtaa tcgacattat atgaatttta</span> 241 <span class="ff_line">actaattccg tttctaattt ataattattt cgttaaacca atcaattccc tttaaacact</span> 301 <span class="ff_line">gcttatgcat attctgtctc aatttatata tggcatgcat cttccgtatt aatttataag</span> 361 <span class="ff_line">ttcattttta ttgatcaagt atttgtggtt ttctttatat aaaaaaatgt attagtgttt</span> 421 <span class="ff_line">ttctgtatta attttataag ttcatcttta tgagaatgct aatgtatttg gacagccaat</span> 481 <span class="ff_line">aaaattccaa gaattgctgc aatcaaagat gaaaccggga ggaaatacta ttgtaatatg</span> 541 <span class="ff_line">gatgtatgca gtggcaacat ggctttgttt tggatccacc tcagggtggt ctttcacatt</span> 601 <span class="ff_line">agaggataac aacatattcc ccaaacaata cccaattata aactttacca cagcgggtgc</span> 661 <span class="ff_line">cactgtgcaa agctacacaa actttatcag agctgttcgc ggtcgtttaa caactggagc</span> 721 <span class="ff_line">tgatgtgaga catgaaatac cagtgttgcc aaacagagtt ggtttgccta taaaccaacg</span> 781 <span class="ff_line">gtttatttta gttgaactct caaatcatgc agagctttct gttacattag ccctggatgt</span> 841 <span class="ff_line">caccaatgca tatgtggtcg gctaccgtgc tggaaatagc gcatatttct ttcatcctga</span> 901 <span class="ff_line">caatcaggaa gatgcagaag caatcactca tcttttcact gatgttcaaa atcgatatac</span> 961 <span class="ff_line">attcgccttt ggtggtaatt atgatagact tgaacaactt gctggtaatc tgagagaaaa</span> 1021 <span class="ff_line">tatcgagttg ggaaatggtc cactagagga ggctatctca gcgctttatt attacagtac</span> 1081 <span class="ff_line">tggtggcact cagcttccaa ctctggctcg ttcctttata atttgcatcc aaatgatttc</span> 1141 <span class="ff_line">agaagcagca agattccaat atattgaggg agaaatgcgc acgagaatta ggtacaaccg</span> 1201 <span class="ff_line">gagatctgca ccagatccta gcgtaattac acttgagaat agttggggga gactttccac</span> 1261 <span class="ff_line">tgcaattcaa gagtctaacc aaggagcctt tgctagtcca attcaactgc aaagacgtaa</span> 1321 <span class="ff_line">tggttccaaa ttcagtgtgt acgatgtgag tatattaatc cctatcatag ctctcatggt</span> 1381 <span class="ff_line">gtatagatgc gcacctccac catcgtcaca gttttctttg cttataaggc cagtggtgcc</span> 1441 <span class="ff_line">aaattttaat gctgatgttt gtatggatcc tgagcccata gtgcgtatcg taggtcgaaa</span> 1501 <span class="ff_line">tggtctatgt gttgatgtta gggatggaag attccacaac ggaaacgcaa tacagttgtg</span> 1561 <span class="ff_line">gccatgcaag tctaatacag atgcaaatca gctctggact ttgaaaagag acaatactat</span> 1621 <span class="ff_line">tcgatctaat ggaaagtgtt taactactta cgggtacagt ccgggagtct atgtgatgat</span> 1681 <span class="ff_line">ctatgattgc aatactgctg caactgatgc cacccgctgg caaatatggg ataatggaac</span> 1741 <span class="ff_line">catcataaat cccagatcta gtctagttt</span></span>*GC% Content for gene:*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
*GC% Content for gene (codon optimized):
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
**